Combined fluorescent in situ hybridization for detection of microRNAs and immunofluorescent labeling for cell-type markers

Identification of the cell type of origin for normal or aberrant gene expression is critical for many studies, and poses a significant problem for some regulatory RNAs such as microRNAs. MicroRNAs are small non-coding RNAs that regulate cellular function by targeting specific mRNAs and reducing the level of their protein product. Aberrant expression of miRNAs in cell-types where they are not normally expressed occurs in several disease conditions. Therefore, it is important to determine not only the expression level of microRNAs, but also where they are expressed. Here we describe a detailed method for fluorescent in situ hybridization (FISH) combined with immunofluorescent labeling for cell-type markers in formalin fixed paraffin embedded (FFPE) sections along with modifications required to adapt the protocol for primary neurons grown in culture. We have combined the specificity and stability of locked nucleic acid (LNA) probes with tyramide signal amplification. To prevent loss of small RNA species, we performed post-fixation with ethylcarbodiimide (EDC). Additionally by omitting protease digestion and using only high temperature with sodium citrate buffer for FFPE sections, we were able to perform immunolabeling for proteins concurrently with in situ hybridization without compromising efficacy of either procedure

MicroRNA-142 reduces monoamine oxidase A expression and activity in neuronal cells by downregulating SIRT1

Aberrant expression of microRNAs (miRs) has been implicated in the pathogenesis of several neurodegenerative disorders. In HIV-associated neurocognitive disorders (HAND), miR-142 was found to be upregulated in neurons and myeloid cells in the brain. We investigated the downstream effects of chronic miR-142 upregulation in neuronal cells by comparing gene expression in stable clones of the human neuroblastoma cell line BE(2)M17 expressing miR-142 to controls. Microarray analysis revealed that miR-142 expression led to a reduction in monoamine oxidase (MAO) A mRNA, which was validated by qRT-PCR. In addition to the mRNA, the MAOA protein level and enzyme activity were also reduced. Examination of primary human neurons revealed that miR-142 expression indeed resulted in a downregulation of MAOA protein level. Although MAOA is not a direct target of miR-142, SIRT1, a key transcriptional upregulator of MAOA is, thus miR-142 downregulation of MAOA expression is indirect. MiR-142 induced decrease in MAOA expression and activity may contribute to the changes in dopaminergic neurotransmission reported in HAND

Combined fluorescent in situ hybridization for detection of microRNAs and immunofluorescent labeling for cell-type markers

Identification of the cell type of origin for normal or aberrant gene expression is critical for many studies, and poses a significant problem for some regulatory RNAs such as microRNAs. MicroRNAs are small non-coding RNAs that regulate cellular function by targeting specific mRNAs and reducing the level of their protein product. Aberrant expression of miRNAs in cell-types where they are not normally expressed occurs in several disease conditions. Therefore, it is important to determine not only the expression level of microRNAs, but also where they are expressed. Here we describe a detailed method for fluorescent in situ hybridization (FISH) combined with immunofluorescent labeling for cell-type markers in formalin fixed paraffin embedded (FFPE) sections along with modifications required to adapt the protocol for primary neurons grown in culture. We have combined the specificity and stability of locked nucleic acid (LNA) probes with tyramide signal amplification. To prevent loss of small RNA species, we performed post-fixation with ethylcarbodiimide (EDC). Additionally by omitting protease digestion and using only high temperature with sodium citrate buffer for FFPE sections, we were able to perform immunolabeling for proteins concurrently with in situ hybridization without compromising efficacy of either procedure

Upregulation of cathepsin D in the caudate nucleus of primates with experimental parkinsonism

<p>Abstract</p> <p>Background</p> <p>In Parkinson's disease there is progressive loss of dopamine containing neurons in the substantia nigra pars compacta. The neuronal damage is not limited to the substantia nigra but progresses to other regions of brain, leading to loss of motor control as well as cognitive abnormalities. The purpose of this study was to examine causes of progressive damage in the caudate nucleus, which plays a major role in motor coordination and cognition, in experimental Parkinson's disease.</p> <p>Results</p> <p>Using chronic 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine treatment of rhesus monkeys to model Parkinson's disease, we found a upregulation of Cathepsin D, a lysosomal aspartic protease, in the caudate nucleus of treated monkeys. Immunofluorescence analysis of caudate nucleus brain tissue showed that the number of lysosomes increased concurrently with the increase in Cathepsin D in neurons. <it>In vitro </it>overexpression of Cathepsin D in a human neuroblastoma cell line led to a significant increase in the number of the lysosomes. Such expression also resulted in extralysosomal Cathepsin D and was accompanied by significant neuronal death associated with caspase activation. We examined apoptotic markers and found a strong correlation of Cathepsin D overexpression to apoptosis.</p> <p>Conclusions</p> <p>Following damage to the substantia nigra resulting in experimental Parkinson's disease, we have identified pathological changes in the caudate nucleus, a likely site of changes leading to the progression of disease. Cathepsin D, implicated in pathogenic mechanisms in other disorders, was increased, and our <it>in vitro </it>studies revealed its overexpression leads to cellular damage and death. This work provides important clues to the progression of Parkinson's, and provides a new target for strategies to ameliorate the progression of this disease.</p

Exosome-mediated shuttling of microRNA-29 regulates HIV Tat and morphine-mediated neuronal dysfunction.

Neuronal damage is a hallmark feature of HIV-associated neurological disorders (HANDs). Opiate drug abuse accelerates the incidence and progression of HAND; however, the mechanisms underlying the potentiation of neuropathogenesis by these drugs remain elusive. Opiates such as morphine have been shown to enhance HIV transactivation protein Tat-mediated toxicity in both human neurons and neuroblastoma cells. In the present study, we demonstrate reduced expression of the tropic factor platelet-derived growth factor (PDGF)-B with a concomitant increase in miR-29b in the basal ganglia region of the brains of morphine-dependent simian immunodeficiency virus (SIV)-infected macaques compared with the SIV-infected controls. In vitro relevance of these findings was corroborated in cultures of astrocytes exposed to morphine and HIV Tat that led to increased release of miR-29b in exosomes. Subsequent treatment of neuronal SH-SY5Y cell line with exosomes from treated astrocytes resulted in decreased expression of PDGF-B, with a concomitant decrease in viability of neurons. Furthermore, it was shown that PDGF-B was a target for miR-29b as evidenced by the fact that binding of miR-29 to the 3\u27-untranslated region of PDGF-B mRNA resulted in its translational repression in SH-SY5Y cells. Understanding the regulation of PDGF-B expression may provide insights into the development of potential therapeutic targets for neuronal loss in HIV-1-infected opiate abusers

Induction of miR-155 after Brain Injury Promotes Type 1 Interferon and has a Neuroprotective Effect.

Traumatic brain injury (TBI) produces profound and lasting neuroinflammation that has both beneficial and detrimental effects. Recent evidence has implicated microRNAs (miRNAs) in the regulation of inflammation both in the periphery and the CNS. We examined the expression of inflammation associated miRNAs in the context of TBI using a mouse controlled cortical impact (CCI) model and found increased levels of miR-21, miR-223 and miR-155 in the hippocampus after CCI. The expression of miR-155 was elevated 9-fold after CCI, an increase confirmed by in situ hybridization (ISH). Interestingly, expression of miR-155 was largely found in neuronal nuclei as evidenced by co-localization with DAPI in MAP2 positive neurons. In miR-155 knock out (KO) mice expression of type I interferons IFNα and IFNβ, as well as IFN regulatory factor 1 and IFN-induced chemokine CXCL10 was decreased after TBI relative to wild type (WT) mice. Unexpectedly, miR-155 KO mice had increased levels of microglial marker Iba1 and increased neuronal degeneration as measured by fluoro-jade C (FJC) staining, suggesting a neuroprotective role for miR-155 in the context of TBI. This work demonstrates a role for miR-155 in regulation of the IFN response and neurodegeneration in the aftermath of TBI. While the presence of neuronal nuclear miRNAs has been described previously, their importance in disease states is relatively unknown. Here, we show evidence of dynamic regulation and pathological function of a nuclear miRNA in TBI

The evolutionary young miR-1290 favors mitotic exit and differentiation of human neural progenitors through altering the cell cycle proteins.

Regulation of cellular proliferation and differentiation during brain development results from processes requiring several regulatory networks to function in synchrony. MicroRNAs are part of this regulatory system. Although many microRNAs are evolutionarily conserved, recent evolution of such regulatory molecules can enable the acquisition of new means of attaining specialized functions. Here we identify and report the novel expression and functions of a human and higher primate-specific microRNA, miR-1290, in neurons. Using human fetal-derived neural progenitors, SH-SY5Y neuroblastoma cell line and H9-ESC-derived neural progenitors (H9-NPC), we found miR-1290 to be upregulated during neuronal differentiation, using microarray, northern blotting and qRT-PCR. We then conducted knockdown and overexpression experiments to look at the functional consequences of perturbed miR-1290 levels. Knockdown of miR-1290 inhibited differentiation and induced proliferation in differentiated neurons; correspondingly, miR-1290 overexpression in progenitors led to a slowing down of the cell cycle and differentiation to neuronal phenotypes. Consequently, we identified that crucial cell cycle proteins were aberrantly changed in expression level. Therefore, we conclude that miR-1290 is required for maintaining neurons in a differentiated state

Traumatic brain injury increases levels of miR-21 in extracellular vesicles: implications for neuroinflammation

Traumatic brain injury (TBI) is an important health concern and effective treatment strategies remain elusive. Understanding the complex multicellular response to TBI may provide new avenues for intervention. In the context of TBI, cell–cell communication is critical. One relatively unexplored form of cell–cell communication in TBI is extracellular vesicles (EVs). These membrane‐bound vesicles can carry many different types of cargo between cells. Recently, miRNA in EVs have been shown to mediate neuroinflammation and neuronal injury. To explore the role of EV‐associated miRNA in TBI, we isolated EVs from the brain of injured mice and controls, purified RNA from brain EVs, and performed miRNA sequencing. We found that the expression of miR‐212 decreased, while miR‐21, miR‐146, miR‐7a, and miR‐7b were significantly increased with injury, with miR‐21 showing the largest change between conditions. The expression of miR‐21 in the brain was primarily localized to neurons near the lesion site. Interestingly, adjacent to these miR‐21‐expressing neurons were activated microglia. The concurrent increase in miR‐21 in EVs with the elevation of miR‐21 in neurons, suggests that miR‐21 is secreted from neurons as potential EV cargo. Thus, this study reveals a new potential mechanism of cell–cell communication not previously described in TBI

A comprehensive study to delineate the role of an extracellular vesicle-associated microRNA-29a in chronic methamphetamine use disorder

Extracellular vesicles (EVs), which express a repertoire of cargo molecules (cf. proteins, microRNA, lipids, etc.), have been garnering a prominent role in the modulation of several cellular processes. Here, using both non-human primate and rodent model systems, we provide evidence that brain-derived EV (BDE) miRNA, miR- 29a-3p (mir-29a), is significantly increased during chronic methamphetamine (MA) exposure. Further, miR-29a levels show significant increase both with drug-seeking and reinstatement in a rat MA self-administration model. We also show that EVassociated miR-29a is enriched in EV pool comprising of small EVs and exomeres and further plays a critical role in MA-induced inflammation and synaptodendritic damage. Furthermore, treatment with the anti-inflammatory drug ibudilast (AV411), which is known to reduce MA relapse, decreased the expression of miR-29a and subsequently attenuated inflammation and rescued synaptodendritic injury. Finally, using plasma fromMUDsubjects, we provide translational evidence that EV-miR29a could potentially serve as a biomarker to detect neuronal damage in humans diagnosed with MA use disorder (MUD). In summary, our work suggests that EVassociated miR-29a-3p plays a crucial role in MUD and might be used as a potential blood-based biomarker for detecting chronic inflammation and synaptic damage

A Comprehensive Study to Delineate the Role of an Extracellular Vesicle-Associated MicroRNA-29a in Chronic Methamphetamine Use Disorder

Extracellular vesicles (EVs), which express a repertoire of cargo molecules (cf. proteins, microRNA, lipids, etc.), have been garnering a prominent role in the modulation of several cellular processes. Here, using both non-human primate and rodent model systems, we provide evidence that brain-derived EV (BDE) miRNA, miR-29a-3p (mir-29a), is significantly increased during chronic methamphetamine (MA) exposure. Further, miR-29a levels show significant increase both with drug-seeking and reinstatement in a rat MA self-administration model. We also show that EV-associated miR-29a is enriched in EV pool comprising of small EVs and exomeres and further plays a critical role in MA-induced inflammation and synaptodendritic damage. Furthermore, treatment with the anti-inflammatory drug ibudilast (AV411), which is known to reduce MA relapse, decreased the expression of miR-29a and subsequently attenuated inflammation and rescued synaptodendritic injury. Finally, using plasma from MUD subjects, we provide translational evidence that EV-miR29a could potentially serve as a biomarker to detect neuronal damage in humans diagnosed with MA use disorder (MUD). In summary, our work suggests that EV-associated miR-29a-3p plays a crucial role in MUD and might be used as a potential blood-based biomarker for detecting chronic inflammation and synaptic damage